How genomic stability is maintained during cell divisions and development is a fundamental question for Cell and Developmental Biology with a strong implication in human diseases including cancer. To maintain a stable genome, cells rely on the concerted action of several cellular processes, including faithful DNA replication, efficient DNA repair, and coordinated cell cycle progression. The DNA damage-signaling pathway, also known as the DNA damage checkpoint, is the central conductor of these processes. The ataxia-telangiectasia mutated and rad3-related (ATR) checkpoint kinase is a master regulator of the checkpoint pathway. As the main cellular sensor of a broad spectrum of DNA damage and genomic instability, ATR plays a key role in protecting the genome, particularly during DNA replication. Compromised ATR signaling increases genomic instability, contributing to the development and progression of cancer. Furthermore, ATR is activated by radiation and many chemotherapeutic drugs, and defective ATR response renders cancer cells highly sensitive to extrinsic and intrinsic DNA damage. Given the pivotal role of ATR in DNA damage response and cancer therapy, it is of great importance to understand the molecular mechanisms by which ATR is activated and by which it functions. A better understanding of the regulation and function of ATR will be critical for the development of new cancer therapies to target the genomic instability in cancer cells. Our main goal in this proposal is to understand how ATR is transformed into a fully active kinase during DNA damage response. Furthermore, we will investigate how the ATR- Chk1 signaling pathway is activated during the early stage of DNA damage response, and how it is deactivated at the late stage of the response. Our studies will combine biochemical, cell biological, and proteomic approaches. In particular, we will analyze how ATR and its regulators and effectors are modified after DNA damage, and how these modifications regulate their functions. We will also systematically develop an in vitro biochemical assay system to recapitulate the activation of ATR by DNA damage. These studies will allow us to clearly define the key molecular events that lead to ATR activation, as well as the key events that orchestrate the signaling through the ATR-Chk1 pathway. These studies may significantly advance the current model of ATR activation, providing the molecular basis for future studies to reveal the full biological functions of ATR and its implications in targeted cancer therapy. PUBLIC HEALTH RELEVANCE: Compromised ATR function leads to increased genomic instability, which has been linked to cancer, developmental defects, and premature aging. Understanding the molecular mechanisms by which ATR is activated and by which it functions will help elucidate the underlying mechanisms of these diseases, and help develop new therapeutic strategies for the treatment of these diseases.